Pgl3 firefly luciferase control vector

The AKT2 and AKT3 3′UTRs containing the predicted miR-29b target sequence were amplified from genomic DNA (A2780 and SKOV3 cells) and cloned into the pGL3 firefly luciferase control vector (Promega, Madison, WI) at the XhoI restriction site immediately downstream of the luciferase reporter gene. To generate AKT2/AKT3 3′UTRs with a mutant target sequence, transversion mutations of 7 nucleotides were made at the miR-29b seed region complementary sites as shown in Figure 3C. Post-transcriptional inhibition of the luciferase reporter gene by miR-29b was assayed in A2780 and SKOV3 cells. Briefly, ovarian cancer cells were seeded into 24-well plates and cultured until 70%–80% confluent. The cells were then co-transfected with either miR-29b or control mimics at a 120 nM final concentration or with 200 ng of pGL3 reporter construct containing wild-type or miR-29b with the mutated AKT2/AKT3 3′UTR using the X-treme GENE HP DNA Transfection Reagent according to the manufacturer's recommendations (Roche, Indianapolis, IN, USA). The transfections were performed in quadruplicate. Relative firefly luciferase activity, which was normalized with Renilla luciferase, was measured 48 h post-transfection using a dual-luciferase reporter gene assay system (Promega, Madison, WI, USA), and the results were plotted as percentage change over the respective control.

Human BRCA2 promoter-silencer (− 921 to + 301) was amplified from genomic DNA isolated from BT549 BC cells using primers P1 and P3 (Additional file 1: Table S1) [13 (link)]. The amplified PCR product was cloned into pCRIV-Topo (Invitrogen) and its sequence was verified using primers T7 and T3. The promoter-silencer insert was digested out of the recombinant plasmid with EcoRI and subcloned into pRL-Null (Promega). Clones with the insert in the reverse orientation with respect to the T7 RNA polymerase promoter (pRL-PS) was selected and used for reporter assays to study the effect of the silencer on BRCA2 promoter activity in SLUG-positive BC cells. Transient transfections were performed in 24-well plates using Lipofectamine 2000 (Invitrogen) with pRL-PS (0.8 μg) and pGL3 firefly luciferase control vector (0.08 μg) (Promega). Protein lysates were prepared from the cells, and luciferase activity was measured as described previously [13 (link)]. Renilla luciferase activity was normalized to the firefly luciferase activity and presented as a ratio (relative light units). Transfected cells were left to recuperate for 6 h before cell synchronization and H₂O₂ treatment. Protein concentrations of the extracts, when needed, were determined using RC-DC reagents and protocol from Bio-Rad.